Flavonol glycosides of berries
Flavonol
glycosides of berries of three major sea huckthorn subspecies, Hippophaë
rhamnoides ssp. rhamnoides, ssp. sinensis and ssp. mongolica
Heikki
Kallioa, Baoru Yanga,b and Teemu Halttunena
aDepartment
of Biochemistry and Food Chemistry, University
of Turku , FIN-20014, Turku , Finland
bAromtech Ltd, Veturitallintie 1,
FIN-95410 Tornio, Finland
Flavonol glycosides of sea buckthorn (Hippophaë
rhamnoides L.) berries were separated by RP-HPLC-DAD analysis and
identified by reference compounds. Glycoside fractions were hydrolyzed by b-glycosidase and the aglycones
identified, again, by HPLC and reference compounds. All the three subspecies investigated (ssp. mongolica,
ssp. rhamnoides, ssp. sinensis) contained the same major
flavonoid glycoside species, the lowest total contents existing regularly in
the mongolica berries. The major aglycon was isorhamnetin followed by
quercetin with minor amounts of kaempferol. The flavonol glycosides, such as
flavonol 3-O-rutinosides, 3-O-glucosides and 3-O-sophoroside-7-rhamnosides were
determined to distinguish between the three subspecies.
Key words: Flavonol glycosides,
Hippophaë rhamnoides, isorhamnetin, kaempherol, quercetin, sea bucktorn
Introduction
Flavonols and flavons protect plants
against UV and visible light and damages caused by bursts of free radicals
(Takahama, 1983). The compounds also have anti-viral and anti-microbial
potential (Parr and Bolwell, 2000). Quercetin seems to be the most common
flavonol in the plant kingdom, being abundant in tee, apples, onion, broccoli
and many other vegetables. In some cases also red wine may have high contents
of flavonols, even up to 50 mg/l (Frankel et al., 1995).
Statistically, quercetin and, in some
extent, also kaempferol were shown to reduce the risk of dying in iscemic heart
disease (Hertog et al., 1995; Knekt et al., 2002). There are also evident indications of
beneficial effects of flavonols on cancer, especially on lung cancer (Stefani
et al., 1999; Knekt et al., 2002).
Materials and methods
Berries
Wild berries of sea buckthorn, Hippophaë
rhamnoides ssp. rhamnoides were picked on Raippaluoto island at
west-coast of Finland, ssp. sinensis in Wenshui county in Shanxi
province in China, and cultivated berries of ssp. mongolica in
Novosibirsk in Russia. The berries were frozen within a day.
Reagents and reference compounds
b-glucosidase was purchased from
Novozyme. The reference compounds isorhmnetin, quercetin, kaempferol,
isorhamnetin 3-O-rutinoside, isorhamnetin 3-O-glucoside, quercetin
3-O-rutinoside and quercetin 3-O-glucoside were from Extrasynthése (Genay,
France) and the internal standard floridzin from Sigma-Aldrich (Steinheim,
Germany). Isorhamnetin 3-O-ß-D-sophoroside-7-O-a-rhamnoside verified by
NMR-analysis (Rösch et al., 2003) was donated by Kroh and Rösch at the
Technical University of Berlin.
Extraction of flavonol glycosides
Methanol and acetic acid (99.8 %) were
added in ratio 14:1 on thawed berry samples (20 g) to reach the total volume of
60 ml. After homogenizing for two minutes the mixture was filtered and the
filtrate was collected. The residue was re-extracted twice with 50 ml of
methanol:water:acetic acid (70:30:5) by homogenizing for one minute and
filtered. After evaporation the residue was dissolved in 25 ml methanol and a
volume of 10 ml of the methanol solution was spiked with 2 ml of the internal
standard, floridzine solution (1.00 mg/ml). The solution was evaporated to
dryness and dissolved in water (10 ml).
Purification of flavonol glycosides
The extract was purified in one-gram
polyamide cartridge activated with methanol (20 ml) and water (60 ml). After
removal of the polar compounds by water, the flavonol glycoside fraction was
collected with 40 ml of methanol. The fraction was evaporated to dryness,
dissolved in methanol, filtrated and stored at – 20 oC. Each berry
sample was extracted and purified in duplicates and analyzed twice with
HPLC-DAD.
HPLC-DAD
analysis of flavonol glycosides
The HPLC instrument consisted of
Shimadzu SIL-10A auto injector, sample cooler, two CTO-10A pumps, CTO-10A
column oven, SPD-M10AVP diode array detector and SCL 10AVP central unit
(Shimadzu Ltd, Kyoto, Japan). A Phenomex Prodigy ODS 5m (3) column (250 x 4.60 mm,
particle size 5 mm) was applied. The effluent consisted of a mixture of
water – tetrahydrofuran (THF) – trifluoroacetic acid (TFA) (98:2:0.1) (solvent
A) and acetonitril (solvent B). The gradient profile used in glycoside analysis
is presented in Table 1. Flow rate of the effluent was 1 ml/min, the detector
wavelengths 270 nm and 370 nm and the volume of injection 10 ml. Identification was based on
co-injections of reference compounds and comparisons of absorption spectra.
Table 1. Gradients used in HPLC-DAD analysis
|
Glycoside
gradient
|
|
Aglycone
gradient
|
||
|
T / min
|
% B
|
|
T / min
|
% B
|
|
0-2
|
15
|
|
0-2
|
10
|
|
2-14
|
15-25
|
|
2-25
|
10-30
|
|
14-19
|
25
|
|
25-35
|
30-50
|
|
19-24
|
25-60
|
|
35-40
|
50-90
|
|
24-28
|
60
|
|
40-45
|
90
|
|
28-30
|
60-90
|
|
45-50
|
90-10
|
|
30-35
|
90
|
|
50-60
|
10
|
|
35-40
|
90-15
|
|
|
|
|
40-50
|
15
|
|
|
|
Fractionation
of flavonol glycosides and enzymatic hydrolysis
The five major glycoside peaks of the
HPLC-DAD analysis were isolated and re-analyzed with the same method to verify
the purity of the fractions. The glycosidic fractions were evaporated to
dryness under dry N2 stream and the residues dissolved in 1 ml of
0.1 M acetate buffer (pH 4.6). A volume of 10 ml of b-glucosidase solution was added
(activity 640 nkat/ml) and the sample was incubated for 5 h at 45 o
C. The sample hydrolyzed was evaporated to dryness under dry N2 and
the aglycones of the residue dissolved in 1 ml of methanol and filtered.
Analysis of flavonol aglycones
The aglycones were analyzed by HPLC as
defined above, but by using the aglycone gradient (Table 1.)
Results and discussion
Figure 1 shows a chromatogram of
flavonoid glycosides of sea buckthorn ssp. rhamnoides
berries. The five major glycosides numbered from 1 to 5, which were taken into
account in the HPLC analyses, did separate well from other peaks.
Identification of the compounds was based on retention times, co-injection with
the reference compounds, DAD-spectra of the glycosides, and HPLC analysis of
the aglycones isolated from the fractionated and enzyme hydrolyzed glycosides.
The DAD-spectra of the flavonol glycosides together with the aglycone analysis
indicated the reasonable purity of all the the five glycoside peaks taken into
account.
The compounds numbered in Figure 1 as
peaks 1 to 5 were identified as isorhamnetin 3-sophoroside-7-rhamnoside,
quercetin 3-O-rutinoside, quercetin 3-O-glucoside, isorhamnetin 3-O-rutinoside
and isorhamnetin 3-O-glucoside, respectively. Trace amounts of kaempferol
glycosides may have been hidden behind the isorhamnetin 3-O-glucoside and
isorhamnetin 3-O-rutinoside peaks.
1
4
6
5
2
3
Figure 1. HPLC-DAD chromatogram of
flavonol glycosides of sea buckthhorn ssp. rhamnoides
berries. 1 = isorhamnetin 3-sophoroside-7-rhamnoside, 2 = quercetin
3-O-rutinoside, 3 = quercetin 3-O-glucoside, 4 = isorhamnetin 3-O-rutinoside, 5
= isorhamnetin 3-O-glucoside and 6 = phloridzin (internal standard)
In order to verify the purity of the
aglycon moieties of the five glycoside peaks, each peak was isolated by the
analytical HPLC column. A thorough investigation with ssp. rhamnoides
was carried out. Each fraction collected was re-analyzed with the normal
procedure to control that the collection had been successful. After enzymatic
hydrolysis, the aglycones were analyzed by HPLC. The glycoside peaks 1, 4 and 5
yielded isorhamnetin, 100 %, 100 % and > 95 %, respectively, and the
glycoside peaks 2 and 3 quercetin, > 98 % and > 95 %, respectively, as
expected.
Floridzine was used as internal standard
in quantitative analysis of the glycosides. Correction factors for the
glycosides in HPLC analysis were 1.0 for isorhamnetin 3-O-rutinoside, 0.70 for
isorhamnetin 3-O-glucoside, 0.90 for quercetin 3-O-rutinoside and 0.72 for
quercetin 3-O-glucoside In case of
isorhamnetin 3-sophoroside-7-rhamnoside, correction factor 1 was applied due to
the lack of the isolated glycoside for the correction factor determination.
Contents of the flavonol glycosides in
different subspecies are summarized in Table 2.
In the subspecies rhamnoides and sinensis the five
compounds were always the most abundant flavonol glycosides. In the berries of
ssp. mongolica isorhamnetin 3-sophoroside-7-rhamnoside existed in trace
amounts only, and some other glycosides not investigated in this study were
found in more abundance. The proportions of unknown flavonol glycosides seen in
Figure 1. could not be defined due to the HPLC-DAD analysis without known
response factors.
Table 2. Content of flavonol glycosides
in sea buckthorn berries in mg / kg and
% of total flavonols (in parentheses).
|
Compound
|
rhamnoides
|
sinensis
|
mongolica
|
|
I-3-sophoroside-
7-rhamnoside
|
252 ± 22
(31,6 ± 1,9)
|
84,4 ± 6,1
(11,1 ± 0,6)
|
17,3 ± 3,3
(4,9± 0,7)
|
|
I-3-rutinoside
|
47,2 ± 3,0
(5,9± 0,2)
|
55,6 ± 3,0
(7,3 ± 0,6)
|
33,6 ± 5,7
(9,4 ± 1,1)
|
|
I-3-glucoside
|
30,2 ± 2,1
(3,8 ± 0,2)
|
44,5 ± 3,0
(5,9 ± 0,3)
|
36,9 ± 5,8
(10,3± 0,8)
|
|
Q-3-rutinoside
|
69,7 ± 5,5
(8,7 ± 0,5)
|
63,9 ± 2,8
(8,4 ± 0,7)
|
23,3 ± 2,9
(6,6 ± 0,9)
|
|
Q-3-glucoside
|
121 ± 8,0
(15,1 ± 0,7)
|
167 ± 6,1
(22,0 ± 2,2)
|
50,1 ± 7,0
(14,1 ± 2,1)
|
|
total
|
799 ± 30
|
763 ± 81
|
362 ± 85
|
The ssp. rhamnoides berries
always containes high amounts of isorhamnetin 3-sophoroside-7-rhamnoside
regardless of the origing and time of harvesting (unpublished results).
As early as sixty years ago Biehlig
showed the existence of isorhamnetin in sea buckthorn berries (Biehlig, 1944),
and in 1963 Friedrich reported isorhamnetin-3-glucoside and
isorhamnetin-3-rutinoside as typical components of the fruit (Friedrich, 1963).
It is of common knowledge that in addition to isorhamnetin and quercetin
glycosides also kaempferol and myricetin
glycosides exist in sea buckthorn berries (Hoerhammer et al., 1966;
Rösch et al, 2003). One of the most reliable investigations is that of Rösch et
al. (2003) where proper NMR-data are presented and e.g. the structure of a
major compound isorhamnetin 3-sophoroside-7-rhamnoside is verified. According
to our knowledge, a comparison between flavonol glycosides of the three,
commercially most important sea buckthorn subspecies, ssp. rhamnoides,
ssp. mongolica and ssp. sinensis has not been published earlier.
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